REVISED ABSTRACT: Type 1 diabetes (T1D) mellitus is a predictable disease and autoantibodies (Ab) against GAD65, IA-2 or insulin (IAA) are effective markers. During the current funding period we have made considerable progress towards the identification of GAD65Ab epitopes associated with T1D. We also established that GAD65Ab were associated with HLA DR3-DQ2 while DR4-DQ8 was associated with IA-2Ab or IAA. IAA were associated with INS VNTR on chromosome 11. We now aim to uncover the mechanisms by which these autoantibody disease markers are triggered and mature in relation to progression towards T1D. The specific aims test these hypotheses:1) Islet autoimmunity is established by the shift towards specific isotype and subtype autoantibodies;2) Progression to T1D is associated with maturation towards specific GAD65Ab epitopes;3) Maturation towards specific autoantibody epitopes is associated with HLA alone, or modulated by non-HLA genetic factors such as INS VNTR for IAA and Oligoadenylate synthetase (OAS) for GAD65Ab. These hypotheses will be tested in prospective studies of large populations of newborns, healthy subjects and studies of school children and first-degree relatives. We will dissect early autoantibody responses including IgM, IgA, and IgE compared to persistent GAD65-lgG using novel high throughput assays with recombinant biotinylated GAD65 and GAD65-specific Fab for epitope mapping. We will a) detect individuals with very early autoantibody response to GAD65, and b) identify isotype switches and maturation to disease-associated epitopes. We will correlate the maturation towards specific autoantibody epitopes with HLA, non-HLA genetic factors, alone or in combination. We will also clarify why some epitope-restricted autoantibodies are associated islet autoimmunity alone and others with progression towards T1D. This research aims to uncover the mechanisms of the early immune response to GAD65 and the relationship between the autoantibody response maturation and progression to clinical onset of T1D.